U.S. patent number 5,837,265 [Application Number 08/612,571] was granted by the patent office on 1998-11-17 for chemically-modified clostridiatoxin with improved properties.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Antonio Ferrer-Montiel, Mauricio Montal.
United States Patent |
5,837,265 |
Montal , et al. |
November 17, 1998 |
Chemically-modified clostridiatoxin with improved properties
Abstract
The invention consists of modified Clostridium neurotoxin
compounds, pharmaceutical compositions containing such compounds
and methods for preparing such compounds. In particular, the
compounds of the invention are purified Clostridium botulinum and
Clostridium tetani neurotoxins in which the tyrosine residues have
been modified to have a negative charge (e.g., by covalent
attachment of a phosphate or sulphate thereto) or in which the
tyrosine residues have been substituted with amino acids having a
negative charge (e.g., glutamate, aspartate, or negatively charged,
non-natural amino acids). Toxins having phosphorylated tyrosine
residues in both the light and heavy chains of the toxins are
preferred. Methods for enzymatic and chemical modification of
tyrosine residues in purified Clostridium neurotoxins are
provided.
Inventors: |
Montal; Mauricio (La Jolla,
CA), Ferrer-Montiel; Antonio (La Jolla, CA) |
Assignee: |
The Regents of the University of
California (Oakland, CA)
|
Family
ID: |
24453738 |
Appl.
No.: |
08/612,571 |
Filed: |
March 8, 1996 |
Current U.S.
Class: |
424/239.1;
424/832; 530/324; 514/18.1 |
Current CPC
Class: |
C07K
14/33 (20130101); A61K 39/08 (20130101); Y02A
50/30 (20180101); A61K 38/00 (20130101); Y10S
424/832 (20130101) |
Current International
Class: |
A61K
39/08 (20060101); C07K 14/195 (20060101); C07K
14/33 (20060101); A61K 38/00 (20060101); A61K
039/00 (); A61K 039/02 (); A61K 039/06 () |
Field of
Search: |
;424/236.1,239.1,247.1
;435/193,194 ;514/2 |
Other References
Database Medline, US National Library of Medicine (Bethesda, MD),
No. 82046912, Shibaeva, et al., "Changes in Biological Properties
of Botulinum Neurotoxin A Induced by Chemical Modification of its
Molecule . . . ". .
Database Embase, Elsevier Sci. B.V., No. 74056271, Stein, et al.,
"Modification of Tetanus Toxin with Selective Chemical Reagents,"
Abstract Z, Immunitatsforsch. Exp. Immun., 1973, vol. 145, No. 5.
.
Database Medline, US National Library of Medicine (Bethesda, MD),
No. 89261699, M. Woody et al., "Effects of Tetranitromethane on the
Biological Activities of Botulinum Neurotixin Types A, B and E",
Abstract. .
Nagahama, et al., "Effect of prior treatment with Clostridium
perfringens epsilon toxin inactivated by various agents on lethal,
pressor and contractile activities . . . ", FEMS Microbiology
Letters 72 (1990) 59-62. .
Robinson, et al., "Tetanus Toxin, The Effect of Chemical
Modifications on Toxicity, Immunogenicity, and Conformation,"
Journal of Biological Chemistry, vol. 250, No. 18, pp. 7435-7442,
Sep. 25, 1975. .
Ferrer-Montiel, et al., "Tyrosine Phosphorylation Modulates the
Activity of Clostridial Neurotoxins," Journal of Biological
Chemistry, vol. 271, No. 31, pp. 18322-18325, Aug. 2, 1996. .
Pernollet, et al., "OH Treatment of Tetanus Toxin Reduced its
Susceptibility to Limited Proteolysis with More Efficient
Presentation to . . . ", Molecular Immunology, vol. 30, No. 18, pp.
1639-1646, 1993. .
Chemical Abstracts, vol. 80, 1974, p. 307, col. 2, abstract No.
25783x, Bizzini et al., "Immunochemistry of Tetanus Toxin.
Nitration of Tyrosyl Residues in Tetanus Toxin.", Eur. J. Biochem.,
1973, vol. 39, No. 1. .
Sakurai et al., "The Inactivation of Clostridium Perfringens
Epsilon Toxin by Treatment with Tetranitromethane and
N-Acetylimidazole," Toxicon, vol. 25, No. 3, pp. 279-284, 1987.
.
Woody et al. Effect of tetranitromethane on biological activities
of botulinum neurotioxin types A,B, and E. Mol. Cell. Biochemistry,
85:159-169, Jan. 1989..
|
Primary Examiner: Tsang; Cecilia J.
Assistant Examiner: Borin; Michael
Attorney, Agent or Firm: Fish & Richardson P.C.
Government Interests
STATEMENT OF FEDERALLY SUPPORTED RESEARCH
The development of this invention was supported by funds provided
by the U.S. Department of the Army, Medical Research Command, under
Grant No. DAMD-17-93-C-3100. The Government may have certain rights
to this invention.
Claims
What is claimed is:
1. A Clostridium neurotoxin having tyrosine residues phosphorylated
to have a negative charge, wherein the proteolytic activity and
thermal stability of the neurotoxin are enhanced as compared to
Clostridium neurotoxins in which negatively charged tyrosine
residues are absent.
2. The neurotoxin according to claim 1 wherein the neurotoxin is a
Clostridium botulinum neurotoxin.
3. The neurotoxin according to claim 2 wherein the neurotoxin is
botulinum toxin A, botulinum toxin B, or botulinum toxin E.
4. The neurotoxin according to claim 1 wherein the neurotoxin is a
Clostridium tetani neurotoxin.
5. A pharmaceutical composition comprising a Clostridium neurotoxin
having tyrosine residues phosphorylated to have a negative charge,
and a pharmaceutically acceptable carrier, wherein the proteolytic
activity and stability of the neurotoxin are enhanced as compared
to Clostridium neurotoxins in which negatively charged tyrosine
residues are absent.
6. The pharmaceutical composition according to claim 5 wherein the
neurotoxin is a Clostridium botulinum neurotoxin.
7. The pharmaceutical composition according to claim 6 wherein the
neurotoxin is botulinum toxin A, botulinum toxin B or botulinum
toxin E.
8. The pharmaceutical composition according to claim 5 wherein the
neurotoxin is a Clostridium tetani neurotoxin.
9. A Clostridium neurotoxin wherein tyrosine residues therein are
sulfated to have a negative charge, wherein further the proteolytic
activity and thermal stability of the neurotoxin are enhanced as
compared to Clostridium neurotoxins in which negatively charged
tyrosine residues are absent.
10. The neurotoxin according to claim 9 wherein the neurotoxin is a
Clostridium botulinum neurotoxin.
11. The neurotoxin according to claim 10 wherein the neurotoxin is
botulinum toxin A, botulinum toxin B, or botulinum toxin E.
12. The neurotoxin according to claim 9 wherein the neurotoxin is a
Clostridium tetani neurotoxin.
13. A pharmaceutical composition comprising a Clostridium
neurotoxin wherein tyrosine residues therein are sulfated to have a
negative charge, and a pharmaceutically acceptable carrier, wherein
further the proteolytic activity and stability of the neurotoxin
are enhanced as compared to Clostridium neurotoxins in which
negatively charged tyrosine residues are absent.
14. The pharmaceutical composition according to claim 13 wherein
the neurotoxin is a Clostridium botulinum neurotoxin.
15. The pharmaceutical composition according to claim 14 wherein
the neurotoxin is botulinum toxin A, botulinum toxin B or botulinum
toxin E.
16. The pharmaceutical composition according to claim 13 wherein
the neurotoxin is a Clostridium tetani neurotoxin.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to pharmacologically active compositions of
neurotoxins obtained from Clostridium botulinum and Clostridium
tetani. Methods for producing the compositions of the invention are
also provided.
2. History of the Invention and Prior Art
The neurotoxin serotypes produced by Clostridium botulinum
(collectively, BoTx) are some of the most potent neuroparalytic
agents known to man and are the causative agents for botulism.
Paradoxically, BoTx serotype A (BoTxA) is also considered to be an
effective pharmaceutical agent for use in the treatment of certain
neuromuscular disorders associated with uncontrolled muscle
contractions or spasms such as disorders of the extraocular muscles
(e.g., strabismus and nystagmus) as well as dystonias (involuntary
contractions of facial, hand and neck muscles) (see, e.g., The New
England Journal of Medicine, 324:1186-1194, 1991).
More recently, BoTxA has been approved for use in temporarily
smoothing facial wrinkles. BoTxA is believed to produce a
reversible, nondegenerative flaccid paralysis of mammalian skeletal
muscle, presumably by blocking the exocytosis of acetylcholine at
peripheral, presynaptic cholinergic terminals, with limited
activity at synapses in the central nervous system (Rabasseda, et
al., Toxicon, 26:329-326, 1988).
Other serotypes of BoTx have been identified that have
immunologically distinct phenotypes; i.e., serotypes B, C1, C2, D,
E, F and G (Simpson, et al., Pharmacol.Rev., 33:155-188, 1981). All
of the BoTx serotypes are believed to be proteins of about 150 kDa
molecular weight that are comprised of two polypeptide chains
linked by disulphide bridges. The shorter of the two chains (the
light chain, LC) is believed to be responsible for the toxicity of
the toxin, while the longer of the two chains (the heavy chain, HC)
is believed to be responsible for the penetration of the toxin into
nervous tissue. Although antigenically different to some extent,
the Botulinum serotypes are believed to be similar in their
pharmacological actions (Brin, et al., "Report of the Therapeutics
and Technology Assessment Subcommittee of the American Academy of
Neurology", Neurology, 40:1332-1336, 1990). For example, each of
the serotypes cleaves cellular protein substrates which are
involved in the release of the acetylcholine neurotransmitter into
the synaptic cleft of neurons in the peripheral cholingergic
nervous system. Protein substrates for BoTx include SNAP-25
(synaptosome-associated protein, cleaved by the A and E serotypes),
syntaxin (cleaved by the C serotype) and synaptobrevin (cleaved by
the B, D, F and G serotypes).
The tetanus toxin produced by Clostridium tetani (TeTx) is believed
to share substantially the same mode of action of BoTx; i.e., TeTx
acts as an anticholinergic, presynaptic neurotoxin. Serotypes A and
E of the BoTx share a substantial degree of sequence homology with
TeTx (DasGupta, et al., Biochemie, 71:1193-1200, 1989). Further,
although TeTx primarily acts on the central (rather than
peripheral) nervous system to produce rigid (rather than flaccid)
muscle paralysis, at least one peptide digestion fragment of TeTx
(fragment Ibc) has been shown to act peripherally to produce
flaccid paralysis in a manner similar to BoTx (Fedinic, et al.,
Boll. Ist. Sieroter Milan, 64:35-41, 1985; and, Gawade, et al.,
Brain Res., 334:139-46, 1985). TeTx cleaves synaptobrevin.
TeTx and most BoTx serotypes are available from commercial sources.
However, as presently manufactured, up to 90% of the active toxin
may be lost during purification to a pharmacologically useful
product, resulting in a composition comprised of a combination of
active and inactive toxin. Typically, a pharmacological purified
toxin composition is lyophilized for storage, then reconstituted
for clinical use with saline or another pharmaceutically acceptable
carrier (see, e.g., the manufacturing and lyophilization process
described in published European Patent Application No. 0 593 176 A2
[process for limiting the volume of inactive toxin in a purified
BoTxA composition]). Once reconstituted, the presently available
toxin compositions are typically unstable and quickly lose potency
at room temperature. Moreover, even when used soon after
reconstitution, the relatively low active toxin concentrations
present in commercially available toxin compositions limit the
activity of each dose, thereby requiring that the toxin be
administered repeatedly over a course of time at the same point of
entry into tissue.
A need, therefore, exists for pharmaceutically acceptable BoTx and
TeTx compounds which have greater thermal stability and proteolytic
activity than do currently available toxin compositions.
SUMMARY OF THE INVENTION
The invention comprises BoTx and TeTx compounds having enhanced
proteolytic activity and thermal stability as compared to toxins
obtained and purified by conventional techniques. Methods and
reagents for use in preparing the toxin compositions of the
invention are also provided.
Specifically, the invention provides pharmaceutically acceptable
compositions of all BoTx serotypes, as well as TeTx compositions.
In each composition, tyrosine residues in at least the LC and,
preferably, in both the LC and HC of the toxin present are
phosphorylated or sulfated to provide a negative charge to each
modified tyrosine. Alternatively, tyrosine residues in the toxins
are substituted with negatively charged amino acids (e.g.,
glutamate, aspartate, or negatively charged non-natural amino
acids). Both L- and D-isomers of each toxin are provided by the
invention. The enhancement of activity and stability of the
inventive toxin compositions as compared to presently available
toxin compositions is illustrated by comparison between fully
phosphorylated BoTxA to BoTxA obtained and purified by conventional
techniques; i.e., unphosphorylated BoTxA.
On contact with a BoTxA substrate (SNAP-25), the substrate is
cleaved by a 10 nanomolar concentration of phosphorylated toxin in
about 5 minutes. In contrast, at even a 50 nanomolar concentration,
cleavage of the substrate is not achieved by the unphosphorylated
toxin until after 30 minutes or more of contact. Further, at a
temperature of 37.degree. C., the unphosphorylated toxin becomes
virtually inactivated within about 2 hours, while the
phosphorylated toxin retains 50% or more of its proteolytic
activity for up to 10 hours at the same temperature. Thus,
modification of the toxin to provide it with a negative charge
enhances the proteolytic activity and thermal stability of the
toxin.
The invention also provides methods for producing BoTx and TeTx
compounds modified according to the invention using either
enzymatic or non-enzymatic (chemical) reagents. Pharmaceutical
compositions containing such compounds are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an autoradiogram depicting phosphorylation of both the
heavy chain (HC) and light chain (LC) of BoTxA by a tyrosine kinase
(pp60.sup.src), measured by incorporation of .sup.32 P into each
chain (from [Y-.sup.32 P] ATP).
FIG. 2 is an autoradiogram depicting pp60.sup.src phosphorylation
of BoTxA, BoTxE, BoTxB and TeTx, measured by incorporation of
.sup.32 P into each chain (from [Y-.sup.32 P] ATP).
FIG. 3 is a graph depicting the rate of phosphorylation of BoTxA by
pp60.sup.src (closed circles) and rate of dephosphorylation of
pp60.sup.src phosphorylated BoTxA in the presence of
protein-tyrosine phosphatase 1B (open circles).
FIG. 4 is a graph depicting the rate of cleavage of SNAP-25 by
pp60.sup.src phosphorylated BoTxA during phosphorylation (closed
circles) and by the same toxin during protein-tyrosine phosphatase
1B dephosphorylation (open circles).
FIGS. 5(a-b) are graphs depicting the rate of cleavage of SNAP-25
by pp60.sup.src phosphorylated BoTxA (closed squares) and
unphosphorylated BoTxA (closed circles). Panel A depicts rate of
cleavage as a function of toxin concentration, while panel B
depicts rate of cleavage as a function of time.
FIGS. 6(a-b) are graphs depicting the rate of cleavage of SNAP-25
by pp60.sup.src phosphorylated BoTxE (closed squares) and
unphosphorylated BoTxE (closed circles). Panel A depicts rate of
cleavage as a function of toxin concentration, while panel B
depicts rate of cleavage as a function of time.
FIG. 7 is a graph depicting the rate of cleavage of SNAP-25 by
pp60.sup.src phosphorylated BoTxA (open squares and circles) and
unphosphorylated BoTxA (closed squares and circles) which were
preincubated prior to the cleavage array at either 22.degree. C.
(squares) or 37.degree. C. (circles).
FIGS. 8(a-b) are graphs depicting the rate of cleavage of SNAP-25
by pp60.sup.src phosphorylated BoTxA (closed squares) and
unphosphorylated BoTxA (closed circles), wherein the
unphosphorylated starting BoTxA materials were inactivated (to a
loss of approximately 90% activity) at 37.degree. C. for 6 hours
before phosphorylation of one-half of the materials and incubation
with SNAP-25.
FIG. 9 is a graph depicting the predicted extent of inhibition of
[.sup.3 H]-noradrenaline neurotransmitter from neuronal (PC12)
cells on incubation of the cells with pp60.sup.src phosphorylated
BoTxE (closed circles) or unphosphorylated BoTxA (open
circles).
FIG. 10 depicts the single channel currents of pp60.sup.src
p-phosphorylated BoTxA HC reconstituted in lipid bilayer
membranes.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. METHODS FOR PRODUCING NEGATIVELY CHARGED, TYROSINE MODIFIED BoTx
AND TeTx
A. Unphosphorylated BoTx and TeTx Starting Materials
Unphosphorylated TeTx and known serotypes of BoTx for use as
starting materials to produce the toxin compositions of the
invention may be purified to homogeneity using techniques which
will be known to, or can be readily ascertained by, those of
ordinary skill in the art. For reference in this regard,
descriptions of each purified BoTx serotype are provided in Shone,
Clostridium botulinum neurotoxins, their structures and modes of
action, "Natural Toxicants in Foods" (Horwood, 1987) at pp 11-57.
The manufacturing method utilized in producing a commercial BoTxA
product supplied by the United Kingdom firm Porton under the
tradename DYSPORT.TM. is set forth at Hambleton, J.Neurol.,
239:16-20 (1992); in addition, a related isolation and purification
technique is described in Hambleton, et al., Production,
purification and toxoiding of Clostridium botulinum type A toxin,
"Biomedical Aspects of Botulism" (Acad. Press, 1981) at pp 247-260.
Reported amino acid sequences and structural characteristics for
the BoTx and TeTx toxins can also be found at, for example, Eisel,
et al., EMBO J., 5:2495-2502, 1986; Hambleton, et al., Botulinum
toxin structure, action and clinical uses, "Neurotoxins and Their
Pharmacological Implications" (Raven Press, 1987) at pp. 233-260
[BoTx toxins]; Thompson, et al., EurJ.Biochem., 189:73-81, 1990
[BoTxA; nucleotide sequence for the encoding gene included];
Montal, et al., FEBS Lett., 313:12-18, 1992 [TeTx and BoTx channel
forming motifs]; DasGupta and Sugiyama, Biochem.Biophys.Res.Comm.,
48:108-112, 1972 [subunit structure common to BoTx serotypes A, B
and E]. A method useful in producing lyophilized BoTxA is also
described in published EPO Application No. 0 593 176 A2. The
disclosures of these references are each incorporated herein by
this reference for the sole purpose of illustrating the state of
knowledge in the art regarding the isolation and purification of
BoTx and TeTx. The products of each isolation and purification
technique known in the art are unphosphorylated toxins.
With reference to the known structural characteristics of BoTx and
TeTx, conventional techniques for isolation and purification of
proteins may be used (in addition to the particular methods
referred to in the references cited above) to obtain substantially
pure unphosphorylated toxin starting materials useful in producing
the toxin compounds of the invention. Briefly, substantially pure
BoTx or TeTx may be obtained through microbial expression, by
synthesis, or by purification means known to those skilled in the
art, such as affinity chromatography. In this regard, the term
"substantially pure", as used herein denotes a protein which is
substantially free of other compounds with which it may normally be
associated in vivo. In the context of the invention, the term
refers to homogenous toxin, which homogenicity is determined by
reference to purity standards known to those of ordinary skill in
the art (e.g., purity sufficient to allow the N-terminal amino acid
sequence of the protein to be obtained).
BoTx and TeTx peptides can be synthesized by such commonly used
methods as t-BOC or Fmoc protection of alpha-amino groups. Both
methods involve stepwise syntheses whereby a single amino acid is
added at each step starting from the C terminus of the peptide
(see, Coligan, et al., Current Protocols in Immunology, Wiley
Interscience, 1991, Unit 9). Peptides of the invention can also be
synthesized by various well known solid phase peptide synthesis
methods, such as those described by Merrifield (J. Am. Chem Soc.,
85:2149, 1962), and Stewart and Young (Solid Phase Peptides
Synthesis, Freeman, San Francisco, 1969, pp 27-62), using a copoly
(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.
On completion of chemical synthesis, the peptides can be
deprotected and cleaved from the polymer by treatment with liquid
HF-10% anisole for about 1/4-1 hours at 0.degree. C. After
evaporation of the reagents, the peptides are extracted from the
polymer with 1% acetic acid solution which is then lyophilized to
yield the crude material. This can normally be purified by such
techniques as gel filtration on a "SEPHADEX G-15" or "SEPHAROSE"
affinity column. Lyophilization of appropriate fractions of the
column will yield the homogeneous peptide or peptide derivatives,
which can then be characterized by such standard techniques as
amino acid analysis, thin layer chromatography, high performance
liquid chromatography, ultraviolet absorption spectroscopy, molar
rotation, solubility, and quantitated by the solid phase Edman
degradation.
Additionally, unphosphorylated BoTx and TeTx toxin preparations may
be obtained from commercial sources. Examples of such sources and
their respective products include BoTxA preparations supplied by
Allergan, Inc. of Irvine, Calif. under the tradename "BOTOX" and by
Porton Products Ltd, of the United Kingdom under the tradename
"DYSPORT". A pentavalent toxoid of all eight known Botulinum
serotypes is also available as an investigational drug from the
U.S. Center for Disease Control in Atlanta, Ga. Preparations of
individual BoTx serotypes are also available from Wacko of Tokyo,
Japan. Of the individual BoTx serotype preparations, BoTxA
preparations are currently the most preferred for their known
safety and efficacy. Tetanus toxins for use as vaccines are also
commercially available from, for example, Lederle Laboratories of
Wayne, N.J. under the tradename "TETANUS TOXOID PUROGENATED" as
well as from Sigma Chemical of St. Louis, Mo. and Calbiochem of San
Diego, Calif.
B. Phosphorylation of BoTx and TeTx
For convenience, this disclosure will refer primarily to the
preferred embodiment of the invention wherein tyrosine residues in
a BoTx or TeTx compound are phosphorylated. However, while the
invention is not limited by any particular theory concerning the
mechanism by which the proteolytic activity and thermal stability
of the toxins is enhanced, phosphorylation of the tyrosine residues
results in a shift in the charge of the toxin product from neutral
to negative. Thus, those of ordinary skill in the art will
appreciate that other modifications of the toxin which produce a
shift in the toxin to a negative charge ("negatively charged"
toxin) at sites where tyrosine residues are present in the
unmodified toxin would also be expected to enhance the activity and
stability of the toxin, including sulfation of the tyrosine
residues (by covalent attachment of a sulphate to the hydroxy group
of tyrosine residues in the toxin) or substitution of all or a
portion of the residues with negatively charged residues (e.g.,
aspartate, glutamate, or non-naturally occurring, negatively
charged amino acids). So modified, such negatively charged toxins
are regarded for purposes of this disclosure as having
"charge-modified" tyrosine residues. In other words, the term
"charge-modified tyrosine residues" shall refer to both negatively
charged tyrosine residues (prepared according to the invention) and
toxins wherein tyrosine residues have been substituted with
glutamate, aspartate, or non-naturally occurring amino acids having
a negative charge.
In the latter respect, it is well recognized that L-bond peptides
are susceptible to proteolytic degradation, restricting their
application as drugs. However, this obstacle has been successfully
bypassed in some cases by synthesizing analogues which contain
D-bond amino acids or non-natural amino acids. The addition of a
single D-amino acid at the C-terminal position is enough to enhance
the resistance to proteolytic degradation by exopeptidases, without
significantly altering the secondary structure of the peptide
[Abiko, supra]. Resistance to endopeptidases can be achieved by
including individual non-cleavable non-peptidic bonds in points in
the peptide sequence that are specially sensitive to enzymatic
degradation [Meyer, et al., J. Med. Chem. 38:3462-3468 (1995);
Guichard, et al., Peptide Research 7:308-321 (1994)]. Reverse amide
bonds .PSI.[NHCO], reduced amide bonds .PSI.[CH.sub.2 NH] or
retro-reduced bonds .PSI.[NHCH.sub.2 ] can be used as surrogates of
the amide link [CONH] in ESUPs of the invention. Reduced amide
links are preferred, since they result only in minor
destabilisation of .alpha.-helices [Dauber-Osguthorpe, et al., Int.
J. Pep. Prot. Res. 38:357-377 (1991)]. Thus, the invention includes
BoTx and TeTx modified to substitute negatively charged,
non-natural amino acids for tyrosine residues in the toxins.
Additionally, charge-modified toxins of the invention can be
synthesized in all-D-conformations. All-D-peptides can be equally
active as the original all-L-peptides [Merrifield, et al., Ciba
Foundation Symposium 186:5-20 (1994); Wade, et al., Proc. Natl.
Acad. Sci. USA 87:4761-4765 (1990)], capable of successfully
resisting enzymatic degradation [Wade, supra; King, et al., J.
Immunol. 153:1124-1131 (1994)] and less immunogenic than their
all-L-analogues [King, supra].
Although not previously known in the art, BoTx serotypes
(particularly, BoTxA, BoTxB and BoTxE) as well as TeTx are
substrates for the tyrosine kinase pp60.sup.src (FIG. 1 and Example
1). pp60.sup.src is a member of the src family of protein kinases
that are known to phosphorylate a focal adhesion kinase and induce
morphological transformations (e.g., rounding and detachment) in
affected cells. pp60.sup.src is also abundant in brain synaptic
vesicles. pp60.sup.src is obtainable by isolation and purification
from native sources (e.g., from brain tissue homogenates) or may be
purchased commercially from sources such as United States
Biochemical of Cleveland, Ohio.
Phosphorylation of tyrosine residues in BoTx and TeTx by
pp60.sup.src is selective (i.e., residues other than tyrosine are
not phosphorylated) and occurs throughout both the LC and HC (FIG.
2 and Example 1). For example, half-maximal phosphorylation of the
tyrosine residues appears to occur within about 30 minutes on
incubation of 250 nanomolar BoTxA starting material with as little
as about 3-6 Units of pp60.sup.src in buffer (FIG. 3 and Example
1). This ratio of starting material to enzyme (about 250 nanomolar:
3-6 Units) is one in which a minimal amount of enzyme may be used
to preserve the enzyme resource and is therefore preferred over
effective, but less efficient, protocols in which larger quantities
of enzyme are utilized. The reaction is reversible (see, FIG. 3 and
Example 1). Similar results are obtainable with another tyrosine
kinase recently discovered in neuronal PC12 cells, PYK2 [Lev, et
al., Nature, 376:737-745 (1995)]. Those of ordinary skill in the
art will be readily able to extrapolate the ratios of starting
material to enzyme provided herein for production of larger batches
of toxin products according to the invention. All ratios of
starting material to enzyme in which a toxin product having
enhanced proteolytic activity and/or thermal stability (as defined
below) is produced are considered to be within the scope of the
invention; in this respect, the amount of enzyme utilized will be
considered to be a "catalytically effective amount" of enzyme.
Tyrosine phosphorylation is a phenomenon which is a key step in
signal transduction pathways mediated by membrane proteins. Thus,
other tyrosine kinases (such as other members of the src kinase
family) can be expected to be suitable for use as phosphorylating
agents in the method of the invention. Surprisingly, however, the
enhancement of toxin activity and stability achieved in the
invention in specific to toxins in which the tyrosine residues have
been modified in vitro to have a negative charge by either covalent
attachment of a phosphate or sulphate to the hydroxy group of such
residues or substitution of tyrosine residues in the toxin with a
negatively charged amino acid (e.g., glutamate or aspartate).
Modification of serine or threonine residues in the toxins does not
affect the toxins' activity or stability. Thus, serine
phosphorylating agents would not be expected to be useful in the
method of the invention.
Although enzymatic phosphorylation is preferred for its
specificity, phosphorylation may also be produced chemically; i.e.,
without reliance on a catalytic reaction. Reagents known to be
useful in phosphorylating tyrosine residues in proteins include
di-t-butyl N,N-diethyl-phosphoriamidite (for phosphorylation in a
t-butyl phosphate protection reaction; see, e.g., Perich and
Reynolds, Int.J.Pept.Protein Res., 37:572-575 (1991) [incorporated
herein by this reference for the purpose of illustrating use of the
specified reagent in a conventional peptide synthesis technique]
and the discussion above concerning peptide synthesis using the
well-known Fmoc/solid phase synthesis technique) and
Fmoc-Tyr(PO.sub.3 Me.sub.2)--OH (also applied in the Fmoc/solid
phase synthesis technique; id.).
For use in substitution of tyrosine residues with glutamate or
aspartate, methods for substituting amino acids in a protein are
well-known in the art. For example, in step-wise synthesis of a
particular toxin, substitution of tyrosine with aspartate and
glutamate may be readily performed by one of ordinary skill in
peptide synthesis. Alternatively, using a toxin-encoding
polynucleotide as a starting material, a desirable mutation may be
produced by site-specific mutagenesis using a conventional
polymerase chain reaction (PCR) and a primer pair corresponding to
the 3' and 5' regions of the cDNA. A preferred method of
mutation-generating PCR amplification is the overlap extension PCR
technique described by Ho, et al., Gene 77:51-59 (1989), the
disclosure of which is incorporated herein by this reference.
Generally, this technique accomplishes site-specific mutagenesis of
the clone by utilizing a 3' primer to add the mismatched mutating
bases (primer B in the Ho article, which is used with the 5' primer
A in the first PCR cycle described). Amplification using the A and
B primers yields an AB fragment. A second PCR cycle uses a primer
(D) from the 3' end of the gene and a 5' mutated primer C
complementary to primer B. The resulting amplification product
(fragment CD) will overlap the AB fragment. When the AB and CD
fragments are denatured, reannealed and amplified using the A and D
primers, the resulting fusion product (AD) will contain the
full-length cDNA sequence and the desired mutation. Another
suitable approach to making single base substitutions or deletions
is described by Shaw in U.S. Pat. No. 4,904,584 ("Site-Specific
Homogenenous Modification of Polypeptides") , the disclosure of
which is incorporated herein by this reference for purposes of
illustrating knowledge in the art regarding methods for achieving
specific mutations in polynucleotides and polypeptides.
Enhancement of the activity and thermal stability of the toxin
products of the inventive process will vary depending on whether
the LC, HC or both are phosphorylated. Preferably, both chains will
be phosphorylated for maximal enhancement of the toxins'
properties, including enhancement of thermal stability (FIG. 7 and
Example 3). In this respect, phosphorylation of the LC results in
enhancement of the toxins' proteolytic properties (see, FIGS. 4
through 6 and Example 2), while phosphorylation of the HC appears
to enhance the toxins' channel gating capabilities by extending the
time in which the membrane channels formed by the HC remain open
for translocation of the LC into the cytosol (see, FIG. 10 and
Example 6). In vivo, phosphorylation of the toxin also appears to
potentiate the toxins' activity in blocking the release of
neurotransmitters into the synaptic cleft (see, FIG. 8 and Example
5), probably by enhancement of the proteolytic activity of the LC.
Given the relatively long-lived activity of BoTx and TeTx observed
in vivo (as compared to the relatively short-lived activity of the
toxins in vitro), it is probable that in situ phosphorylation of
the toxin protects it from intracellular degradation. Thus, in
vitro phosphorylation of tyrosine residues likely confers similar
protection against toxin degradation both in vitro and in vivo, as
evidenced by enhancement of the toxins' in vivo activity and in
vitro stability.
Selective enhancement of either the channel gating or proteolytic
activities of the toxin is achievable by phosphorylating only the
HC or LC, respectively. Selective phosphorylation of either chain
of BoTx or TeTx is performed by cleaving the HC and LC chains for
phosphorylation of the tyrosine residues on only the chain to be
phosphorylated or by phosphorylating both chains, cleaving the
chains and reversing phosphorylation on one chain by applying a
dephosphorylating agent such as protein-tyrosine phosphatase 1B
(FIG. 4 and Example 2). Compositions containing mixtures of the
phosphorylated and unphosphorylated chains are prepared as
described below with respect to the fully phosphorylated
toxins.
Quantitatively phosphorylation of both chains of BoTx or TeTx
enhances the proteolytic activity of the toxin by 50% or more
compared to the unphosphorylated toxin (FIGS. 4 through 6 and
Example 2). However, "enhancement" of toxin proteolytic activity
within the meaning of the invention (i.e., as a result of providing
a negative charge at the sites of tyrosine residues in the toxin
LC) will be considered to occur when the concentration of
negatively charged toxin required to produce the same catalytic
reaction in vitro in the same time as the unphosphorylated toxin is
detectably reduced; e.g., by at least about 10%. Thus, the scope of
the invention encompasses phosphorylation of BoTx and TeTx (and the
resulting phosphorylated product) using reagents of varying
phosphorylating efficiencies, so long as a demonstrable enhancement
of the toxin's proteolytic activity as compared to the
unphosphorylated toxin occurs.
The thermal stability of the phosphorylated toxin is also enhanced
as compared to the unphosphorylated toxin. In particular, BoTx or
TeTx having both chains phosphorylated according to the invention
can be expected to retain its proteolytic activity in saline at
room temperature for at least 5 times longer than the
unphosphorylated toxin (FIG. 7 and Example 4). However,
"enhancement" of toxin stability within the meaning of the
invention (i.e., as a result of providing a negative charge at the
sites of tyrosine residues in the toxin) will be considered to
occur when retention of the proteolytic activity of the negatively
charged toxin in saline at room temperature (37.degree. C.)
compared to the unphosphorylated toxin is detectably increased;
e.g., by at least about 10%. Thus, the scope of the invention
encompasses negatively charged BoTx and TeTx (and the resulting
products) using reagents of varying efficiencies, so long as a
demonstrable enhancement of the toxin's stability as compared to
the unphosphorylated toxin occurs.
Interestingly, tyrosine phosphorylation of BoTx and TeTx LC has the
unexpected effect of restoring proteolytic activity of
unphosphorylated toxins which have become inactivated; e.g., after
excessive exposure to moderate heat (see, FIG. 8 and Example 5).
Thus, the methods of the invention for preparation of toxins having
charge-modified tyrosine residues is useful both in producing
pharmaceutically acceptable BoTx and TeTx compositions having
enhanced activity as well as restoring activity to conventional
unphosphorylated BoTx and TeTx compositions after inactivation.
Assays for use in confirming enhancement of the proteolytic
activity and stability of BoTx and TeTx prepared according to the
invention are described in the Examples appended hereto. Other
techniques for measuring the proteolytic activity and stability of
proteins will be well-known to those of ordinary skill in the art
and can be readily practiced without undue experimentation (see,
e.g., Methods in Enzymology (Acad. Press, 1981), the disclosure of
which is incorporated herein by reference only to illustrate the
state of knowledge in the art concerning techniques for testing and
measuring catalytic activity in enzymes). Such assays will be
useful not only in determining whether modification of the charge
of a particular toxin has enhanced its activity or stability within
the meaning of the invention, but will also be useful in
determining whether a particular modification of the charge
adversely affects the secondary structure and catalytic activity of
the toxin to aid in selection and design of pharmaceutically useful
negatively charged toxins having charge-modified tyrosine residues
according to the invention.
C. Pharmaceutically Acceptable BoTx and TeTx Compositions
Charge-modified BoTx and TeTx compositions of the invention are
prepared by mixing the charge-modified toxin having the desired
degree of purity with physiologically acceptable carriers. Such
carriers will be nontoxic to recipients at the dosages and
concentrations employed. Ordinarily, the preparation of such
compositions entails combining the particular protein with buffers,
antioxidants such as ascorbic acid, low molecular weight (less than
about 10 residues) polypeptides, proteins, amino acids,
carbohydrates including glucose or dextrins, chelating agents such
as EDTA, glutathione and other stabilizers and excipients.
Such compositions may be lyophilized for storage and will be
reconstituted according to pharmaceutically acceptable means; i.e.,
suitably prepared and approved for use in the desired application.
A sodium chloride free buffer is preferred for use as a
reconstituting agent. Inclusion of bovine or human serum albumin
(BSA or HSA) in the composition or reconstituting agent has also
been reported to assist in recovery of toxin activity after
reconstitution from a lyophilized state (see, e.g., published EPO
application No. 0 593 176 A2). Whatever its form, the composition
product will be placed into sterile containers (e.g., ampules) for
storage and transportation.
Examples illustrating the practice of the invention are set forth
below. These examples should not be regarded as limiting the scope
of the invention, which is defined by the appended claims. Standard
abbreviations (e.g., "ml" for milliliters, "h" for hours) are used
throughout the examples.
EXAMPLE I
ENZYMATIC PHOSPHORYLATION OF BoTx AND TeTx TYROSINES BY
pp60.sup.src
Using 250 nanomolar quantities of each toxin, the phosphorylation
reaction was conducted in a final volume of 20-40 .mu.l containing
20 mM MgCl.sub.2, 1 mM EGTA, 1 mM DTT, 20 mM Hepes (pH 7.4), 3-6
Units of tyrosine kinase pp60.sup.src, 0.1 mM ATP and 4 .mu.Ci
[.sup.32 P]-.gamma.-ATP (3,000 Ci/mmol; Amersham). Reactions
proceed at 30.degree. C. at 10 minute intervals for a total of 120
minutes and were terminated by addition of 200 .mu.M peptide
A(pp60.sup.v-src [137-157] [from Peninsula], a specific inhibitor
of pp60.sup.src). For comparison regarding the specificity of
tyrosine phosphorylation by pp60.sup.src, BoTxA was incubated with,
separately, serine kinases bovine heart PKA (4 Units, Sigma) and
rat brain PKC(20 ng, UBI). For PKC, the phosphorylation buffer was:
20 mM Hepes, pH7.4, 0.1 mM CaCl.sub.2, 10 mM MgCl.sub.2, 0.25 mg/ml
of L-.alpha.-phosphatidyl-L-serine and 1 mM DTT.
To prepare autoradiograms to detect phosphorylation of each toxin,
PKC and PKA, phosphorylated samples were subjected to SDS-PAGE on
12% gels. Gels were stained with Coomassie blue R-250, destained,
dried and exposed to Kodak X-Omat AR.TM. x-ray film. For
immunoblots, protein bands were electrotransferred onto
nitrocellulose membranes. Bands were visualized using the ECL
radiographic system (Amersham). Control samples included samples
from which kinases had been omitted. For pp60.sup.src, an
additional control sample was preincubated with peptide A.
Purified BoTxA was strongly phosphorylated by pp60.sup.src but not
by the serine kinases PKA or PKC. The autoradiograms display
incorporation of .sup.32 P into both LC (M.sub.r .about.50 kD) and
HC (M.sub.r .about.100 kD) (FIG. 1). pp60.sup.src also
phosphorylates the HC and LC of BoTxA, BoTxB, BoTxE and TeTx.
Autoradiograms display .sup.32 P incorporation into protein (FIG.
2).
As shown in FIG. 3, the phosphorylation reaction between
pp60.sup.src, BoTx and TeTx is reversible. To determine whether the
reaction was reversible, dephosphorylation was conducted in vitro
in a final volume of 15 .mu.l containing 0.1 .mu.M
tyrosine-phosphorylated BoTxA and 50 ng PTP-1B-agarose bead
conjugate. Reactions proceed at 37.degree. C. for indicated times,
and were terminated by centrifugation (14,000 rpm, 1 min) of the
PTP-agarose bead conjugate. The extent of BoTxA dephosphorylation
was assessed by SDS-PAGE. Controls included omission of PTP or
inhibition of PTP with 200 .mu.M VO.sub.4. Phosphorylation of BoTxA
was reversed under these conditions.
EXAMPLE II
ENHANCEMENT OF PROTEOLYTIC ACTIVITY IN THE TOXINS OF THE
INVENTION
Augmentation of BoTxA protease activity by protein tyrosine
phosphorylation. a. Extent of .sup.32 P incorporation into BoTxA HC
and LC as function of incubation time in presence of Src. BoTxA was
incubated with Src at 30.degree. C. for the indicated time periods,
as described in FIG. 1. The band with M.sub.r .about.50 kD
corresponds to Src, which is autophosphorylated. b.
Tyrosine-phosphorylated BoTxA is enzymatically dephosphorylated by
PTP-1B. c. The extent of phosphorylation (a) and dephosphorylation
(b) were quantified using an image analyzer. d. Tyrosine
phosphorylation of BoTxA is accompanied by augmentation of protease
activity. Fluorograms display the mobility change of in vitro
translated SNAP-25 due to proteolysis as function of
phosphorylation time by Src. Last lane shows a control experiment
in which BoTxA was omitted. e.
To measure the proteolytic activity of toxins modified as described
in Example I, and to compare that activity to the activity of
unmodified toxins, the time required for the modified and
unmodified toxins to cleave their respective substrates was tested
and compared at various toxin concentrations. To this end, in vitro
translation of the cDNA clone coding for SNAP-25 from mouse brain
in the presence of [35S]methionine was performed with a
transcription-translation-coupled reticulocyte lysate system
(Promega). The concentration of modified and unmodified BoTxA or
BoTxE incubated with samples of SNAP-25 as shown in FIG. 4 was 10
nM and varied as shown in FIGS. 5 and 6; cleavage reactions were
performed at 30.degree. C. In FIG. 4, the unmodified toxin was
dephosphorylated as described in Example 1 and the effect of
dephosphorylation measured as a function of a decline in
proteolytic activity. In FIG. 5, the unmodified toxin was a
conventional preparation of BoTxA. In FIG. 6, the unmodified toxin
was a conventional preparation of BoTxE.
Samples were analyzed by SDS-PAGE followed by fluorographic
detection of .sup.35 S-labeled proteins. The protein band with
M.sub.r .about.25 kD is illustrated in cleavage was indicated by a
shift to lower M.sub.r. Fluorograms were quantified using the NIH
program Image 1.57. Two selection windows corresponding to
uncleaved SNAP-25 (u) and uncleaved plus cleaved SNAP-25 (u+c) were
defined. The integrated density (ID) of each selection was given by
ID=N(Mean-Background), where N was the number of pixels in the
selection, and the background was the modal gray value (most common
pixel value) after smoothing the histogram. The extent of SNAP-25
cleavage (%), is then given by: (1-ID.sub.u /ID.sub.u+c X100). The
resulting data was converted to graph form for ease of
comparison.
As shown in FIG. 4, dephosphorylation of BoTxA as described in
Example I resulted in a decline of proteolytic activity by a
magnitude of about three times (5 minutes cleavage rate for the
modified toxin (closed circles) versus about 15 minutes cleavage
time for the dephosphorylated toxin (open circles)). As shown in
FIG. 5, the modified BoTxA (closed squares) displayed as much as
50% greater proteolytic activity than the unmodified toxin (closed
circles; Panel A displays the results obtained as a function of
toxin concentration while Panel B displays the results as a
function of time). As shown in FIG. 6, the enhancement of activity
on the part of modified BoTxE (closed squares) as compared to
unmodified BoTxE (closed circles) was similar to the enhancement
measured in modified BoTxA (Panel A displays the results as a
function of toxin concentration while Panel B displays the results
as a function of time).
EXAMPLE III
ENHANCEMENT OF THERMAL STABILITY IN THE TOXINS OF THE INVENTION
Augmentation of BoTx protease activity by protein tyrosine
phosphorylation is accompanied by increased thermal stability (FIG.
7). This enhancement in thermal stability was determined by
measuring the rates of cleavage of in vitro translated SNAP-25 by
tyrosine phosphorylated BoTxA (open squares and circles) or
unphosphorylated BoTxA (closed squares and circles) which were
preincubated at either 22.degree. C. (squares) or 37.degree.
(circles) prior to the cleavage assay. The assay was conducted at
30.degree. C. at a fixed toxin concentration of 20 nM.
EXAMPLE IV
RESTORATION OF ACTIVITY TO THERMALLY INACTIVATED TOXINS BY
MODIFICATION OF THE TOXIN ACCORDING TO THE INVENTION
To determine whether activity could be restored to thermally
inactivated toxins by modifying the toxins according to the
invention, unmodified BoTxA was thermally inactivated to a loss of
approximately 90% of its original activity by exposure to room
temperature (37.degree. C.) for 6 hours. One-half of the
inactivated materials were tyrosine phosphorylated as described in
Example I. The modified and unmodified toxin samples were incubated
with SNAP-25 as described in Example II. As shown in FIG. 8, the
modified toxin displayed proteolytic activity similar in magnitude
to the modified toxins described in Example III (closed squares)
while the unmodified toxin displayed little proteolytic activity
(closed circles).
EXAMPLE V
INHIBITION OF NEUROTRANSMITTER RELEASE: PREDICTED ENHANCEMENT OF
INHIBITORY ACTIVITY IN THE TOXINS OF THE INVENTION
Intracellular tyrosine phosphorylation of BoTxA LC in
NGF-differentiated PC12 cells is associated with potentiation of
neurotransmitter release blockade. To predict the effect of
modification of the toxins of the invention on their inhibitory
activity with respect to neurotransmitter release, pheochromocytoma
(PC12) cells were propagated in RPMI 1640 medium, supplemented with
10% donor horse serum-5% bovine fetal serum, at 37.degree. C. in a
humidified incubator with 5% CO2. For NGF (nerve growth factor)
cellular differentiation (50 ng/ml, for 4 days), PC12 cells were
plated at a density of 10.sup.6 cells/ml on 75 cm.sup.2 culture
dishes with rat tail collagen.
Cells were incubated with modified or unmodified BoTxA dissolved in
PBS for 12 h. Cells were stimulated with PBS supplemented with
indicated additives, washed with cold PBS, lysed with cold
radioimmunoprecipitation (RIPA) buffer (20 mM Tris-HCl pH 7.4, 200
mM NaCl, 1 mM NaF, 1 .mu.g/ml pepstatine A and 1 mM
phenylmethylsulfluoride), incubated and shaken at 4.degree. C.
Insoluble material was removed by centrifugation at 10,000.times.g
for 30 min at 4.degree. C. Soluble material was analyzed by
immunoblot or used for immunoprecipitation. Tyrosine-phosphorylated
proteins were immunoprecipitated with an antiphosphotoyrosine
monoclonal antibody. Immunocomplexes were captured with
agarose-conjugated protein G (Pierce), and analyzed by SDS-PAGE and
immunoblotting. Bands were visualized using the ECL system
(Amersham) to confirm incorporation of the toxins into the
cells.
The cells were stimulated with nerve growth factor and the extent
of [.sup.3 H]-labeled noradrenaline neurotransmitter release from
the cells measured. While the results of these experiments are
preliminary, as shown in FIG. 9, release of noradrenaline is
predicted by the data to be inhibited to a substantially greater
degree in the cells treated with modified BoTxA (closed circles) as
compared to the cells treated with unmodified BoTxA (open
circles).
EXAMPLE VI
ENHANCEMENT OF CHANNEL GATING ACTIVITY IN THE TOXINS OF THE
INVENTION
To determine the non-channel activity of the toxins of the
invention, modified BoTxA were reconstituted into lipid bilayer
membranes. As shown in FIG. 10, the channel formed by the modified
toxin exhibits a high probability of being in the open state (top
current level). This indicates augmentation of the ion channel
activity of the modified toxin HC in comparison to unmodified
toxin.
The invention having been fully described, modifications thereof
will become apparent to those of ordinary skill in the art. All
such modifications of the invention are to be considered to be
within the scope of the appended claims.
* * * * *